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Rheumatol IntDOI 10.1007/s00296-014-2978-2

ORIgInal aRtIcle

Effects of 12‑week combined exercise therapy on oxidative stress in female fibromyalgia patients

Banu Sarıfakıoglu · Aliye Yıldırım Güzelant · Eda Çelik Güzel · Savas Güzel · Ali Rıza Kızıler

Received: 26 July 2013 / accepted: 25 February 2014 © Springer-Verlag Berlin Heidelberg 2014

over a 12-week period. all parameters were reevaluated after the treatment in the patient group. the oxidative stress parameters levels were significantly higher, and antioxidant parameters were significantly lower in patients with fibro-myalgia than in the controls. VaS, FIQ, and BDI scores decreased significantly with exercise therapy. the exercise improved all parameters of oxidative stress and antioxidant parameters. also, all clinical parameters were improved with exercise. We should focus on oxidative stress in the treatment for fibromyalgia with the main objective of reducing oxidative load.

Keywords Fibromyalgia · Oxidative stress · antioxidant status · exercise

Introduction

Fibromyalgia (FM) is a syndrome characterized by chronic muscle pain and tenderness. It is accompanied by somatic and psychological symptoms such as impaired concen-tration, poor memory, sleep disturbance, anxiety, and depression [1] and is the most frequent cause of muscu-loskeletal pain in young and middle-aged women [2]. the etiology and pathogenesis of the disease remain unknown [1], although recent studies have emphasized oxidative stress as a possible cause [3–7].

Free radicals, namely reactive oxygen species (ROS) or reactive nitrogen species (RnS), are the main source of oxi-dative stress in living organisms [8]. their formation in the body is prevented through antioxidant systems, and cellular injury is inhibited by their inactivation [9]. Oxidative stress shifts the dynamic redox balance on behalf of the oxidative potential between oxidant and antioxidant systems [10]. ROS can damage Dna, proteins, and lipids and can cause

Abstract the aims of this study were to investigate the effect of exercise therapy on the oxidative stress in fibro-myalgia patients and relationship between oxidative stress and fibromyalgia symptoms. thirty women diagnosed with fibromyalgia according to the american college of Rheumatology preliminary criteria, and 23 healthy women whose age- and weight-matched women were enrolled the study. Pain intensity with visual analog scale (VaS), the number of tender points, the fibromyalgia impact ques-tionnaire (FIQ), the Beck depression inventory (BDI) were evaluated. the oxidative stress parameters thiobarbitu-ric acid reactive substances, protein carbonyls, and nitric oxide, and antioxidant parameters thiols and catalase were investigated in patients and control group. after, combined aerobic and strengthen exercise regimen was given to fibro-myalgia group. exercise therapy consisted of a warming period of 10 min, aerobic exercises period of 20 min, mus-cle strengthening exercises for 20 min, and 10 min cooling down period. therapy was lasting 1 h three times per week

B. Sarıfakıoglu (*) · a. Y. güzelant Department of Physical Medicine and Rehabilitation, School of Medicine, namik Kemal University, 100, Yıl Mah, Barbaros cad. Merkez, tekirdag, turkeye-mail: banusarifakioglu@yahoo.com

e. Ç. güzel Department of Family Medicine, School of Medicine, namik Kemal University, tekirdag, turkey

S. güzel Department of Biochemistry, School of Medicine, namik Kemal University, tekirdag, turkey

a. R. Kızıler Department of Biophysics, School of Medicine, namik Kemal University, tekirdag, turkey

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lipid peroxidation, a serial reaction whereby electrons are ‘stolen’ from the lipids in cell membranes [11].

carbonyl stress is induced by ROS and involves the release of free carbonyl groups from plasma proteins, such as albumin and lipoproteins, and an increase in plasma free carbonyls. Oxidative stress, which forms part of carbonyl stress, is an important biomarker of protein and tissue dam-age. In proteins, oxidation causes the formation of pro-tein carbonyl groups (Pcgs) [12, 13] and the loss of pro-tein thiol (t-SH) groups [13]. the transformation of t-SH groups to disulfides and other oxidized derivatives, such as oxyacids, is the earliest observable symptom of ROS-medi-ated protein damage [14]. Pcgs are used in protein oxida-tion [15], t-SH level measurement provides antioxidant capacity [16].

nitric oxide (nO) is an unstable messenger molecule and participates in the modulation of pain, vasodilatation, and immune function. During exercise, it acts as a meta-bolic regulator [17] and it is thought to play a role in the modulation of pain in FM [18]. nO levels, relative to those of oxygen, affect lipid peroxidation—if the level of nO is less than or equal to that of oxygen, it is thought to increase lipid peroxidation; if it is higher than that of oxygen, it is thought to reduce lipid peroxidation [19].

the destructive effects of ROS are controlled by enzy-matic (e.g., catalase (cat)) and non-enzymatic (e.g., t-SH) antioxidants [20]; antioxidants eliminate pro-oxidants and scavenge free radicals. It is difficult to quantify ROS and RnS due to their highly reactive properties. therefore, measuring the affected lipid, protein, or nucleic acid prod-ucts may provide the required information. Measurement of lipid peroxidation products (thiobarbituric acid reactive substances —tBaRS), carbonyl groups, and total nitrites and nitrates indicates the degree of lipid peroxidation, pro-tein oxidative injury, and radical formation to total nitric oxide, respectively [13, 21–23].

Patient education, cognitive behavioral therapy, pharma-cotherapy, good sleeping patterns, and combined exercise are recommended for the treatment for FM. In particular, exercise has favorable effects on physical fitness, aerobic performance, pain management, and quality of life of FM patients [24]. the positive effects of regular exercise on oxidative stress and mobility are well established; it is clear that people feel better through physical activity. Some stud-ies have been showed that oxidative stress is higher in FM patients [3, 4, 6], but physical exercise yet was not investi-gated on oxidative status of these patients, so there is insuf-ficient information on this subject.

this study aims to compare the oxidative status of FM patients with respect to normal healthy controls; therefore, the oxidative stress parameters tBaRS, Pcgs, and nO, and antioxidant parameters t-SH and cat were inves-tigated. additionally, was investigated the influence of a

12-week combined aerobic and strengthening exercise pro-gram on oxidative status and clinical symptoms of fibromy-algia patients.

Methods

Patient selection

ethical approval for the study was obtained from the eth-ics committee of the namık Kemal University. Written informed consent was obtained from all study participants prior to the study.

Inclusion criteria, according to revised american col-lege of Rheumatology (acR) preliminary diagnostic cri-teria [25], were diagnosis as primary FM, female sex, and acceptance to participate in the study. Patients with sec-ondary FM (thyroid disease, connective tissue disease) or any condition interfering with exercise (advanced cardiac respiratory or orthopedic problems), those who had under-gone any physical therapy or exercise program within the 6 months prior to the study, those administered any anti-oxidant or antidepressant medication, or smokers, were excluded from the study. thirty-eight patients diagnosed with primary FM and who had a sedentary life included in the study. a patient has undergone a traffic accident, one patient developed plantar fasciitis and six patients not wanted to go on the study. In total, 30 patients were included in the study. Patients were asked not to use any antioxidant medications, such as vitamins c or e, and coenzyme Q10, for the study duration. the control group was composed of 23 healthy, age-matched, non-smoker, and had sedentary lifestyle women.

Body mass index, age, and demographic characteristics were recorded for all patients and controls, and all under-went a general physical examination by the same investiga-tor. tenderness was evaluated by applying pressure (4 kg/cm2) over 18 specific body points, and the number of ten-der points was recorded. FM patients filled visual analog scales (VaS) to evaluate the pain intensity before and after the exercise treatment. Pain was evaluated using a 10-point graded scale; 0 indicated absence of symptoms and 10 indi-cated the worst symptoms. In a valid and reliable turkish version of Beck Depression Inventory (BDI) [26] and a FM Impact Questionnaire (FIQ) [27] were evaluated before and after the exercise treatment.

exercise therapy

exercise therapy, lasting 1 h three times per week over a 12-week period, was performed under supervision by the same physiotherapist (gY). It consisted of a warming period of 10 min, including range of motion (ROM) to all

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joints and slow ambulation, followed by aerobic exercise consisting of 20 min running on a treadmill. the target heart rate was initially adjusted to 65–70 % of the maxi-mal heart rate and to 75–80 % of the maximal heart rate in the advanced program [28]. Muscle strengthening exercises were then performed for 20 min, where deltoid, latissimus dorsi, pectoralis major, abdominalis, gluteus, and quadri-ceps were strengthened through shoulder press, dumb-bell press, shoulder elevation with resistance, biceps curl, squats, hip flexion and extension, and standing hip exer-cises using specified weight loads ranged from 1 to 3 kg weights and two sets of 8–10 repetitions. Finally, relaxa-tion exercises were done in a 10 min cooling down period. Details of the exercise program are summarized in table 1.

Blood samples

Patient and control blood samples (heparinized and non-heparinized) were drawn by venipuncture into pre-cooled tubes and centrifuged at 1,500×g at 4 °c for 10 min. Serum and plasma samples were stored at −70 °c until analysis.

Determination of thiobarbituric acid reactive substance (tBaRS) levels

tBaRS was determined spectrophotometrically by the method of Buege and aust [29]. One volume of plasma was mixed thoroughly with two volumes of 15 % w/v trichlo-roacetic acid, 0.375 % w/v thiobarbituric acid, and 0.25 M Hcl stock solution. the mixture was heated for 30 min in a boiling water bath. after cooling, the flocculent precipitate was removed by centrifugation at 1,000×g for 10 min. the light absorbance of the sample was determined at 535 nm, and tBaRS concentration was calculated using 1.56 × 105 M−1 cm−1 as the molar extinction coefficient.

Determination of protein carbonyl levels

Protein carbonyl levels were determined using 2,4-dinitro-phenyl hydrazine (DnPH), from the difference in absorb-ance at 365 nm between DnPH-treated samples and Hcl-treated controls, with ε370 = 2.000 M−1 cm−1. carbonyl

levels were expressed as nmol of DnPH incorporated/mg of protein [30].

Determination of catalase (cat) activity

goth’s spectrophotometric method [31] was used for the determination of cat activity in serum. the yellow molyb-date and H2O2 complex was measured at 405 nm against a blank. One unit of cat decomposes 1 μmol of H2O2/l min, and results are expressed as kU/l.

Determination of nitric oxide (nO) levels

Serum nO was measured as its stable metabolites nitrate (nO3) and nitrite (nO2). nO3 was first reduced by nitrate reductase to nO2 and then nO2 was determined spectro-photometrically by the griess reaction [32]. griess rea-gent, the (1:1) mixture of 0.2 % naphthalene-diamine and 2 % sulfonamide in 5 % phosphoric acid, gives a red–vio-let diazo dye with nO2 with absorbance at 550 nm. the nO2 + nO3 values are given in μM.

Determination of thiol (t-SH) levels

a spectrophotometric assay based on the t-SH/disulfide reaction of t-SH and ellman’s reagent (5,5′-dithiobis-2-nitrobenzoic acid) was used for the t-SH assay [33]. the t-SH group concentration was calculated using a molar extinction coefficient of 13.600 M−1 cm−1. t-SH levels were expressed as μmol/l.

Statistical analyses

analysis was performed using SPSS for Windows 11.5 package program. normal distribution of continuous vari-ables was assessed by applying the Shapiro–Wilk test, and data were expressed as mean ± standard deviation or as medians and 95 % central range, as appropriate. the dif-ferences between fibromyalgia and control groups were assessed by using unpaired t tests for parametric data and Mann–Whitney U test for nonparametric data. Depend-ent variables were assessed by using paired t tests for

Table 1 Patients were administered 12-week exercise program

ROM range of motion, THR target heart rate, MHR maximum heart rate

type of exercise the exercises time (min)

Warming period Joints (neck, shoulders, elbows, wrists, hips, knees, ankles, low back) ROM exercises 10

aerobic Running exercise at treadmill (starting with tHR, MHRs 65, 75 % and go on to 80 and) 20

Muscle strengthening exercises Shoulder press, dumbbell press, shoulder elevation with resistance, biceps curl, squats, hip flexion and extension and standing hip (8–10 repetitions, 2 sets)

20

cooling down Relaxing exercises 10

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parametric data and Wilcoxon test for nonparametric data to compare fibromyalgia group before and after the exer-cise program. correlations between variables were evalu-ated using Spearman’s correlation coefficient. Odds ratios are presented with 95 % confidence intervals; p values of less than 0.05 were considered statistically significant.

Results

there were no significant differences in age and BMI between the patient and control groups. However, there was a significant difference in oxidative stress parameters between patient and control groups and oxidant load capac-ity of antioxidants was higher in the patient group. Results are shown in table 2.

the clinical assessment of patients with VaS, FIQ, and BDI revealed significant differences before and after exer-cise; all three clinical parameters significantly improved with exercise (table 3). Oxidative stress parameters, tBaRS, nO, and Pcg, showed a significant difference before and after exercise; there was a significant decrease in all three parameters following the 12-week exercise regime. t-SH and cat levels also showed a significant difference before and after treatment, both parameters increased significantly with treatment (table 3).

Before the exercise regime, Pcg was positively cor-related with tBaRS (r = 0.561; p < 0.001) and nO (r = 0.571; p < 0.001). Pcg was negatively correlated with t-SH (r = −0.466; p < 0.001) and cat (r = −0.522; p < 0.001). cat had a positive correlation with t-SH

(r = 0.324; p = 0.018). there was a negative correlation between nO and cat (r = −0.470; p < 0.001) and nO and t-SH (r = −0.328; p = 0.017). When we looked into the clinical parameters, there was a positive correlation between FIQ and BDI (r = 0.475; p = 0.008) and between FIQ and VaS (r = 0.905; p < 0.001). Similarly, there was a positive correlation between BDI and VaS (r = 0.424; p = 0.020). after exercise, there was a positive correlation between Pcg and nO (r = 0.372; p = 0.043). there was a positive correlation between FIQ and BDI (r = 0.598; p < 0.001) and between FIQ and VaS (r = 0.932; p < 0.001). Similarly, there was a positive correlation between BDI and VaS (r = 0.602; p < 0.001).

Discussion

FM is characterized by diffuse pain and tenderness accom-panied by many other symptoms. the pathophysiology of FM is still unclear, although genetic and environmental fac-tors are emphasized [34]. Recently, it has been proposed that oxidative stress may play a role in the etiopathogenesis of FM [3–6]. However, this has not yet been clearly identi-fied. Whether FM is indeed an oxidative stress disease must first be established.

In a study by altindag et al. [4], total peroxide levels were shown to be increased in FM patients; and chung et al. [34] did not observe a significant difference between 48 FM cases and the control group when measuring F2-iso-prostane in urine to evaluate oxidant stress. Bagis et al. [3] investigated malondialdehyde (MDa) levels as an indica-tor of lipid peroxidation in FM patients and found a signifi-cant increase with respect to the control group. likewise, Ozgocmen et al. [7] found a higher tBaRS level in 30 FM

Table 2 Demographic findings, clinical and oxidative status of FM and control group

FM fibromyalgia, BMI Body Mass Index, TP tender point, WPI Widespread Pain Index, SSS Symptom Severity Scale Score, TBARS thiobarbituric acid reactive substance, T-SH thiol, NO nitric oxide

FM group (n = 30)

control group (n = 23)

p

age (year) 42.3 ± 7.82 44.08 ± 8.90 0.450

BMI (kg/m2) 28.12 ± 4.69 28.27 ± 4.15 0.899

Duration of symptoms (months)

65.80 ± 53.49

tP 16.2 ± 1.39

WPI 14.9 ± 3.48

SSS 8.86 ± 1.99

tBaRS (μM) 4.21 ± 0.94 3.29 ± 0.87 0.001

t-SH(μM) 288.28 ± 45.12 401.70 ± 85.96 0.001

Protein carbonyl (mmol/mg)

1.34 ± 0.37 0.76 ± 0.24 0.001

nO 39.49 ± 7.43 28.53 ± 8.10 0.001

catalase (kU/l) 41.03 ± 9.03 52.65 ± 11.84 0.001

Table 3 clinical parameters and oxidative status in FM patients before and after exercise therapy

FM fibromyalgia, Group 1 before exercise therapy, Group 2 after exercise therapy, VAS Visual analog Scale, FIQ Fibromyalgia Impact Questionnaire, BDI beck depression inventory, TBARS thiobarbituric acid reactive substance, T-SH thiol, NO nitric oxide

FM group p

group 1 (n = 30) group 2 (n = 30)

VaS 6.33 ± 1.64 4.93 ± 1.43 0.001

FIQ 58.24 ± 16.25 49.12 ± 7.65 0.001

BDI 24.60 ± 13.33 21.16 ± 12.24 0.001

tBaRS (μM) 4.21 ± 0.94 3.24 ± 0.60 0.001

t-SH(μM) 288.28 ± 45.12 341.41 ± 55.01 0.001

Protein carbonyl (mmol/mg)

1.34 ± 0.37 1.10 ± 0.31 0.001

nO 39.49 ± 7.43 30.73 ± 4.51 0.001

catalase (kU/l) 41.03 ± 9.03 51.35 ± 12.45 0.001

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patients compared with the control group. However, eisen-ger et al. [6] determined that blood plasma MDa levels were unchanged. In a study, it was shown that, Pcg was increased in 25 female FM patients compared with the con-trol group, indicating increased protein peroxidation [35]. Studies of nO levels in patients with FM have also revealed different results; some have demonstrated a decrease in plasma nO levels compared with the control groups [36, 37] (plasma nitrite levels, total antioxidant status, total oxidant status, and oxidative stress index in patients with tension-type headache and fibromyalgia), whereas another study did not observe a significant difference between the two groups [7].

there are contradictory results regarding the decrease in antioxidant capacity in FM patients. two studies found a low total antioxidant status in FM patients [4, 37]. Ozgoc-men et al. [7] used superoxide dismutase (SOD) activities for evaluation of antioxidant status and found that there was no significant difference between the FM and control groups. However, Bagis et al. [3] reported that SOD activ-ity was significantly lower in FM patients than in the con-trol group. Similarly, Sendur et al. [8] showed that cat activity in FM patients was significantly lower than in the control group. One study investigating t-SH levels to eval-uate antioxidant capacity found them significantly lower in FM patients compared with the control groups [38].

In this study, tBaRS, Pcg, and nO levels were meas-ured to show oxidant status, and t-SH and cat activities were used to show antioxidant status. the results demon-strated a deterioration of the balance between oxidant and antioxidant status in female FM patients compared with the control group and the presence of increased oxidative stress in FM patients. Some studies advocate that oxidative stress may cause peripheral and central sensitization, may change the nociception, and may cause hyperalgesia [39]. therefore, we should focus on oxidative stress in the treat-ment for FM with the main objective of reducing oxidative load.

Regular exercise induces biochemical reactions for reducing the harmful effects of ROS. Mammalian skeletal muscle is capable of adapting quickly to exercise through the remodeling of gene expression, cellular structures, and up-regulation of cellular defense mechanisms [40]. the repetition of the same type of exercise induces an adaptive mechanism termed ‘repeated bout effect’ in both humans and animals [41]. Repeated exercise reduces oxidative stress and increases antioxidant capacity. Ilhan et al. [42] showed that recurrent intense exercise programs reduced lipid peroxidation products and decreased oxidative stress through the activation of antioxidant systems.

the results of the present study showed a significant decrease in oxidative stress parameters and improvement of clinical data in FM patients following the 12-week

combined exercise program; thus, indicating that exercise therapy can significantly reduce oxidative load. to our knowledge, this is the first study to evaluate the relation-ship between exercise therapy and oxidative stress param-eters and to show a significant decrease in oxidative stress parameters among women with FM.

FM patients are individuals with low aerobic tolerance and restricted flexibility and strength. It is well-known that exercise is an effective treatment for muscle pain; there-fore, it is a treatment modality with an ever increasing significance for FM patients [43]. the european league against Rheumatism (eUlaR) also recommends aerobic and strengthening exercises in FM patients [44]. It has been shown that strengthening and aerobic exercises reduced pain and tenderness [45]. However, the mechanism of action of exercise treatment in these patients has not been established. the effect of exercise is multifactorial, includ-ing central and peripheral mechanisms. Structural changes in muscle fibers, impaired breakdown in capillary micro-circulation, mitochondrial derangement, and impairment of tissue oxygenation were observed in FM patients [29, 46]. Muscle oxygenation increases through exercise, lead-ing to decreased peripheral and central sensitization and improvement of clinical pain [47]. (Physical exercise also improved the frequently observed symptoms of low mood status, depression, anxiety, and impaired life quality in FM patients. By our study, we showed the decrease in oxidant load with exercise. So leading us to think that the wellness produced by exercise treatment in FM patients depends on decrease in oxidative stress parameters.

Oxidative stress might be associated with FM symp-toms. lipid peroxidation may play a role in depression; peroxidation may reduce the effect of selective serotonin reuptake inhibitors [48]. Further, it is advocated that eryth-rocyte MDa levels correlate with Hamilton depression rating scale (HDRS) resulting in FM [7]. cipollone et al. [49] have found a significant correlation between increased oxidative stress and fatigue; Richard et al. [50] observed a relationship between oxidative stress and fatigue, but not with pain and tenderness. In the present study, a significant improvement was detected in the post-exercise measure-ments through FIQ, BDI, and VaS. exercise therapy was shown to reduce the burden of oxidant with a significant improvement in the clinical data, which suggests that clini-cal findings are linked to the oxidant load.

Most patients enrolled in the study were able to toler-ate the treatment, only six patients dropped out. If the FM patients were motivated and encouraged well for the treat-ment although their symptoms, they can show improve-ment in quality of life by adapting them to exercise therapy. therefore, exercise therapy should be recommended in these patients and patients should be motivated and encour-aged in this regard.

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the study limitations include a small cohort size despite the significance of the measurements, the lack of long-term output evaluation to observe the duration of oxidative stress parameters and symptom relief, and the lack of variation in the exercise protocols to observe the effect of particular exercises on oxidative capacity in order to obtain the best exercise protocol.

although the oxidative stress parameters in FM patients have been previously investigated, this is the first study demonstrating the decrease in these parameters through exercise treatment. In FM patients, a regular exercise regi-men should be recommended and encouraged. Further studies including a larger patient cohort and with differ-ent exercise subgroups will provide more detailed results with regards to the best exercise regime to reduce oxidative stress.

Conflict of interest none.

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